Phase separation and molecular ordering of the prion-like domain of the thermosensory protein EARLY FLOWERING 3

Abstract

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compart-mentalisation of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis tha-liana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical and structural techniques to investigate the dilute and con-densed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher order oligomer that does not depend on the pres-ence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive man-ner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force mi-croscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These ex-periments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Heterotypic interactions in the dilute phase can drive co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex

“…S5 ). Our previous studies have reported that BRG1, the catalytic subunit of the mSWI/SNF complex, can enrich within optogenetically induced FUS PLD condensates and FUS fusion protein condensates in live cells 63–65 . We posited that this functional engagement can be, in part, mediated by disordered PLDs of FUS and BRG1.…”

“…Apart from tyrosine residues, ARID1A PLD primary sequence shows enrichment of glutamines in the C-terminal region with multiple polyQ tracts with stretches of three to four Gln residues (Table S4). Previous studies have suggested that polyQ regions can promote LCD self-association 62,63 . However, when we mutated these Gln residues to Gly and created an ARID1A PLD variant, termed ARID1A PLD 30QtoG , we observed only a modest (~2 fold) increase intracellular Csat (Figs.…”

Tracing the evolutionary emergence of the temperature sensing prion-like domain in EARLY FLOWERING 3 across the plant kingdom

“…Arabidopsis thaliana ELF3 is known to harbor a prion-like domain (PrLD) which is required for phase separation of ELF3 in response to temperature changes (Jung et al, 2020; Hutin et al, 2023). The study of mammal and especially yeast prions resulted in a various prediction tools for PrLDs that are based on two different models.…”

“…Sensing changes in ambient temperature is the first step in plant thermomorphogenesis. Among the known plant temperature sensors, the PrLD in Arabidopsis thaliana ELF3 mediates liquid-liquid phase separation to form aggregates at elevated temperatures (Jung et al, 2020; Hutin et al, 2023). However, it is unknown whether and how the PrLD is conserved in ELF3 orthologs across the plant kingdom.…”

Molecular mechanisms underlying coordinated responses of plants to shade and environmental stresses

“…The evening complex, a protein complex composed of EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRYTHMO (LUX), links the circadian clock to the diurnal control of hypocotyl elongation and flowering (Nusinow et al., 2011; Zhao et al., 2021). Recently, ELF3 was shown to serve as a thermosensor through liquid–liquid phase separation in response to warm temperatures, which is driven by its prion‐like domain (PrLD) (Hutin et al., 2023; Jung et al., 2020) (Figure 2). Under normal temperatures, the evening complex directly binds to the promoters of PIF4 and PIF5 and represses their expression (Nusinow et al., 2011).…”